We plan to study enzymatic activities involved in DNA replication that are affected by diadenosine 5', 5'''-P1,P4-tetraphosphate (Ap4A). Ap4A is formed in the back reaction of the first step of protein synthesis, from ATP and aminoacyl adenylates, and was shown to be directly related to the proliferative activities of mammalian cells. Recently we have shown that the nuclear compartment pools of ATP and nuclear ATP/ADP ratios act as S phase controls. DNA replication in isolated nuclei in vitro, was inhibited by high ATP levels and high ATP/ADP ratios. Studies of intact cells in vivo, were consistent with the in vitro results - although the total cellular pools of ATP and ATP/ADP ratios increase upon progression of mammalian cells through the G1 and into the S phase of their cycle, the nuclear compartment pools of adenine nucleotides and nuclear ATP/ADP ratios decrease upon entry into S phase of the cell cycle. Preliminary experiments have demonstrated that two nuclear enzymatic activities--ATPase and AMP deaminase increase substantially upon the entry of mammalian cells into the S phase of their cycle. These enzymatic activities are responsible for the in vivo changes in the nuclear compartment pools of adenine nucleotides. We plan to study the allosteric effects of Ap4A, ATP and ADP on enzymatic activities that are associated with proteins of the replication complex. Studies of regulatory mechanisms will be conducted on synchronized intact and permeabilized cells as well as isolated nuclei. Interaction of adenine nucleotides with proteins and their effect on enzymatic activities involved in DNA replication will be studied with multiprotein aggregates, as well as with purified proteins.